Project I investigates embryonic stem cell pluripotency directly through the generation of chimeric primate blastocysts, fetuses and offspring with nonhuman primate ES cells (nhpES) - including nhpES derived from blastocysts generated from nuclear transfer (NTnhpES). Through sophisticated non-invasive imaging, this project responsibly answers crucial questions about: primate ES pluripotency during development (aim I);primate ES cell stability and utility after NT (aim II);genomic imprinting in NHPs to solve clinically urgent concerns regarding ART epigenetic consequences; and primate ES fate after transplantation (aim IV). Nine questions are posed in four Specific Aims: Aim I. To dynamically image nhpES cell contributions in developing primate chimerae: 1.1. Will nhpES cells contribute to chimeric blastocysts, fetuses and healthy offspring? 1.2.With tetraploid embryos (4N), will ES cells contribute primarily to the inner cell mass in chimeric blastocysts, fetus and offspring? Primate chimera are generated in four ways, and the fate of each chimera is followed in vitro during preimplantation development, determining the cellular contributions of the ES and tetraploids to the expanded blastocyst stage after differentially labeling ES or embryos with GFP-transgenes, as well as in utero during fetal development and in the offspring. Aim II. Are ES cells derived after nuclear transfer (NTnhpES cells) developmentally restricted? Two questions are posed: 2.1. Are NTnhpES cell lines stable? 2.2 Will NTnhpES cells contribute to chimeric fetuses and offspring? Aim III. Do Genomic Imprints in NHP Embryos, Fetuses, Placentae, Amniotic Cells and Offspring Differ between ART and NT-Chimerae versus Natural Matings? 3.1. What is the level of DNA methylation in embryos, fetuses, placentae, and offspring after ART, NT, or natural matings? 3.2. What are the parental expressions of selected imprinted genes during NHP preimplantation development? 3.3. What is the DNA methylation status of specific genes in rhesus preimplantation embryos? Aim IV. Dynamic imaging of ES cell fates after transplantation. 4.1. Are NT-derived nhp-ESCs tolerated when transplanted back into the female from which both the somatic cell and the oocyte were obtained? 4.2 Are allogenic ES cells rejected like allografts or do their pre-implantation origins confer immunological privilege? This aim will investigate the tolerance of nhpES in allografts and autografts. Taken together, this project will provide crucial information regarding ES cell pluripotency in nonhuman primates and evaluate the safety of stem cell transplantation in nonhuman primates.